*2.8. RNA Labelling, Amplification, Hybridization, and Data Analysis*

First, 200 ng of total RNA were converted to cDNA using oligodT primer tagged to T7 polymerase promoter at 40 ◦C. cDNA thus obtained was converted to cRNA using T7 RNA polymerase enzyme. The dye Cy3 was also incorporated during this step. Labeled cRNA was then cleaned using Qiagen

RNeasy Mini kit columns (Qiagen, Cat No: 74106, Hilden, Germany) and quality assessment was carried out using the Nanodrop ND-1000. Following this, Cy3-labeled cRNA was fragmented at 60 ◦C. Fragmented cRNA was hybridized on the array (AMADID: 027511) using the Gene Expression Hybridization kit (Agilent Technologies, Santa Clara, CA, USA) at 65 ◦C for 16 h in Sure hybridization Chambers. Hybridized slides were washed using Agilent Gene Expression wash buffers (Agilent Technologies, Santa Clara, CA, USA) and scanned on an Agilent Microarray Scanner (Agilent Technologies, Part Number G2600D). Images thus obtained were quantified using Agilent's Feature Extraction Software Version-10.7 (Santa Clara, CA, USA). Feature-extracted raw data were analyzed using the GeneSpring GX12.6.1 microarray data and pathway analysis tool (Santa Clara, CA, USA). Quartile (75th percentile) normalization was performed. Storey and bootstrapping analysis was performed for multiple testing corrections. The expression profile of K133AS-R parasites was extrapolated on a chromosome map of *Leishmania* parasites using custom R programs. The modulated expression of genes was identified using two criteria: (a) statistical and (b) biological. Statistical significance was determined by the t-test (unpaired) and a *p* value < 0.05 was considered as significant for both K133WT and K133AS-R parasites. The biological cutoff for up- or downregulation was 2-fold. Differentially regulated genes were analyzed for functional classification using the GeneDB, BLAST2GO, and AmiGO databases. The pathway analysis was carried out using the gene Spring GX12.6.7 (Santa Clara, CA, USA) and KEGG pathway analysis tool (Bethesda, MD, USA). Interacting partners of up- or downregulated genes in K133AS-R parasites were identified using the String 9.01 database [24].
